Bottom Line:
Structural models of Cys-loop receptors based on homology with the Torpedo marmorata nicotinic acetylcholine receptor infer the existence of cytoplasmic portals within the conduction pathway framed by helical amphipathic regions (termed membrane-associated (MA) helices) of adjacent intracellular M3-M4 loops.Numerous residues, prominently those at the 435, 436, 439, and 440 positions, were found to markedly influence γ.This approach yielded a functional map of the 5-HT3A receptor portals, which agrees well with the homology model.

Affiliation: From the Division of Neuroscience, Medical Research and Medical Education Institutes, Ninewells Hospital and Medical School, University of Dundee, Dundee DD1 9SY, Scotland, United Kingdom.

ABSTRACTStructural models of Cys-loop receptors based on homology with the Torpedo marmorata nicotinic acetylcholine receptor infer the existence of cytoplasmic portals within the conduction pathway framed by helical amphipathic regions (termed membrane-associated (MA) helices) of adjacent intracellular M3-M4 loops. Consistent with these models, two arginine residues (Arg(436) and Arg(440)) within the MA helix of 5-hydroxytryptamine type 3A (5-HT3A) receptors act singularly as rate-limiting determinants of single-channel conductance (γ). However, there is little conservation in primary amino acid sequences across the cytoplasmic loops of Cys-loop receptors, limiting confidence in the fidelity of this particular aspect of the 5-HT3A receptor model. We probed the majority of residues within the MA helix of the human 5-HT3A subunit using alanine- and arginine-scanning mutagenesis and the substituted cysteine accessibility method to determine their relative influences upon γ. Numerous residues, prominently those at the 435, 436, 439, and 440 positions, were found to markedly influence γ. This approach yielded a functional map of the 5-HT3A receptor portals, which agrees well with the homology model.

Figure 6: The influences of arginine substitution and modification of engineered cysteine substitution by MTS reagents upon the single-channel conductance (γ) of the 5-HT3A(QDA) receptor.A, plot depicting the strong correlation (r2 = 0.89) between the change in γ produced by arginine substitution and by the reaction of substituted cysteine residues by positively charged MTSEA at residues 431–441, inclusive, both compared with alanine. B, inclusion of residues 426–430 at which the presence of arginine was associated with an increase in conductance versus alanine controls weakens the correlation (r2 = 0.58). C, correlation (r2 = 0.68) between the magnitude of the increase in γ produced by challenge with MTSES versus the γ of the construct without exposure to the MTS reagent.

Mentions:
For hydrophilic MTS reagents to react with a substituted cysteine residue, the latter must reside in an accessible aqueous environment (51). The positively charged aminoethyl moiety donated to a cysteine residue by MTSEA results in a side chain with a volume and charge similar to that of an arginine residue, although the latter is considerably more basic. Nevertheless, qualitative similarities between the effects of arginine substitutions and covalent modification of cysteine residues by MTSEA would be anticipated for those cysteine residues that are accessible. Application of MTSEA (200 μm) within the patch pipette typically resulted in a decreased γ when compared with the matched alanine controls. The suppression of γ was significant for receptors containing the F428C, K431C, E434C, I435C, D436C, V438C, A439C, and A440C mutations, the greatest decrease being associated with modification of cysteine residues at positions 435 (46%), 436 (45%), and 440 (50%) (Fig. 5A, Table 1). However, such a comparison includes the effect upon γ of the cysteine substitution itself, which in three instances (see above) was significant. Thus, we also analyzed the effect of MTSEA application employing the γ of unmodified cysteine constructs as a reference to isolate the change in γ specifically due to the reagent (Fig. 5B). Using this criterion, MTSEA caused significant reductions in γ at positions 434 through 440 inclusive, in addition to 428 and 431. Notably, the introduction of arginine residues at positions 431 through 440 also caused significant reductions in γ in comparison with alanine controls (see above). At all other loci, MTSEA treatment was associated with a trend toward a reduction in γ. Moreover, the effects upon γ of arginine substitution and challenge with MTSEA at positions 431 through 441 were in excellent quantitative agreement (r2 = 0.89), the data being fitted by a line of regression with a slope (1.13) close to unity (Fig. 6A). However, although arginine substitution at residues 426 through 430 tended to increase γ in comparison with the alanine controls, MTSEA tended to produce the opposite effect. Inclusion of the data obtained for these residues resulted in a much poorer correlation between the effect of arginine substitution or treatment with MTSEA upon γ (r2 = 0.58; Fig. 6B).

Figure 6: The influences of arginine substitution and modification of engineered cysteine substitution by MTS reagents upon the single-channel conductance (γ) of the 5-HT3A(QDA) receptor.A, plot depicting the strong correlation (r2 = 0.89) between the change in γ produced by arginine substitution and by the reaction of substituted cysteine residues by positively charged MTSEA at residues 431–441, inclusive, both compared with alanine. B, inclusion of residues 426–430 at which the presence of arginine was associated with an increase in conductance versus alanine controls weakens the correlation (r2 = 0.58). C, correlation (r2 = 0.68) between the magnitude of the increase in γ produced by challenge with MTSES versus the γ of the construct without exposure to the MTS reagent.

Mentions:
For hydrophilic MTS reagents to react with a substituted cysteine residue, the latter must reside in an accessible aqueous environment (51). The positively charged aminoethyl moiety donated to a cysteine residue by MTSEA results in a side chain with a volume and charge similar to that of an arginine residue, although the latter is considerably more basic. Nevertheless, qualitative similarities between the effects of arginine substitutions and covalent modification of cysteine residues by MTSEA would be anticipated for those cysteine residues that are accessible. Application of MTSEA (200 μm) within the patch pipette typically resulted in a decreased γ when compared with the matched alanine controls. The suppression of γ was significant for receptors containing the F428C, K431C, E434C, I435C, D436C, V438C, A439C, and A440C mutations, the greatest decrease being associated with modification of cysteine residues at positions 435 (46%), 436 (45%), and 440 (50%) (Fig. 5A, Table 1). However, such a comparison includes the effect upon γ of the cysteine substitution itself, which in three instances (see above) was significant. Thus, we also analyzed the effect of MTSEA application employing the γ of unmodified cysteine constructs as a reference to isolate the change in γ specifically due to the reagent (Fig. 5B). Using this criterion, MTSEA caused significant reductions in γ at positions 434 through 440 inclusive, in addition to 428 and 431. Notably, the introduction of arginine residues at positions 431 through 440 also caused significant reductions in γ in comparison with alanine controls (see above). At all other loci, MTSEA treatment was associated with a trend toward a reduction in γ. Moreover, the effects upon γ of arginine substitution and challenge with MTSEA at positions 431 through 441 were in excellent quantitative agreement (r2 = 0.89), the data being fitted by a line of regression with a slope (1.13) close to unity (Fig. 6A). However, although arginine substitution at residues 426 through 430 tended to increase γ in comparison with the alanine controls, MTSEA tended to produce the opposite effect. Inclusion of the data obtained for these residues resulted in a much poorer correlation between the effect of arginine substitution or treatment with MTSEA upon γ (r2 = 0.58; Fig. 6B).

Bottom Line:
Structural models of Cys-loop receptors based on homology with the Torpedo marmorata nicotinic acetylcholine receptor infer the existence of cytoplasmic portals within the conduction pathway framed by helical amphipathic regions (termed membrane-associated (MA) helices) of adjacent intracellular M3-M4 loops.Numerous residues, prominently those at the 435, 436, 439, and 440 positions, were found to markedly influence γ.This approach yielded a functional map of the 5-HT3A receptor portals, which agrees well with the homology model.

Affiliation:
From the Division of Neuroscience, Medical Research and Medical Education Institutes, Ninewells Hospital and Medical School, University of Dundee, Dundee DD1 9SY, Scotland, United Kingdom.

ABSTRACTStructural models of Cys-loop receptors based on homology with the Torpedo marmorata nicotinic acetylcholine receptor infer the existence of cytoplasmic portals within the conduction pathway framed by helical amphipathic regions (termed membrane-associated (MA) helices) of adjacent intracellular M3-M4 loops. Consistent with these models, two arginine residues (Arg(436) and Arg(440)) within the MA helix of 5-hydroxytryptamine type 3A (5-HT3A) receptors act singularly as rate-limiting determinants of single-channel conductance (γ). However, there is little conservation in primary amino acid sequences across the cytoplasmic loops of Cys-loop receptors, limiting confidence in the fidelity of this particular aspect of the 5-HT3A receptor model. We probed the majority of residues within the MA helix of the human 5-HT3A subunit using alanine- and arginine-scanning mutagenesis and the substituted cysteine accessibility method to determine their relative influences upon γ. Numerous residues, prominently those at the 435, 436, 439, and 440 positions, were found to markedly influence γ. This approach yielded a functional map of the 5-HT3A receptor portals, which agrees well with the homology model.